Protein misfolding is one of the nation's most prominent causes of disease and ultimately death. These include neurodegenerative diseases such as Alzheimer's and Parkinson's, heart failure, diabetes, metabolic syndromes and autoimmune diseases. A prominent component of the onset of these diseases is the age dependent decline in protein homeostasis that protects us from proteotoxicity or misfolding and loss of function when we are young. Protein homeostasis, or proteostasis, is normally maintained by regulatory pathways and a network that modulates intracellular concentrations of chaperones, protein degradation machinery like the proteasome, and disaggregation activities, among others. It is crucial to understand how the over 300 proteins involved in protein homeostasis interact to overcome cellular stresses caused by protein aggregation and/or misfolding and excessive degradation. The protein homeostasis network in mammalian cells expressing amyloidgenic or misfolding prone proteins will be characterized and compared to control cell lines. This will be accomplished initially utilizing RNA microarray analysis to monitor transcriptional changes, mass spectrometry-based proteomics to quantify protein level changes, and activity-based enzyme profiling to monitor the activity of chaperones and folding enzymes that consume ATP, activities that may or may not be reflected by their protein levels. The mechanisms of action of recently discovered proteostasis regulators that restore protein homeostasis will be characterized utilizing these approaches as well. This approach should enable us to understand the innate cellular response to proteostasis stresses and the mechanism by which proteostasis regulators restore cellular protein homeostasis to ameliorate protein misfolding diseases. I also aspire to begin to understand the mechanism(s) of cellular proteotoxicity exhibited by amyloidgenic proteins. The toxicity of the functional Pmel17 amyloid and the pathologic A[unreadable] amyloid in non-native cellular compartments will be evaluated, including localization to the cytoplasm, nucleus, endoplasmic reticulum, and the extracellular space of mammalian cells. The response of the proteostasis network to the process of amyloidgenesis in a given compartment will be analyzed as described above. The effects of proteostasis regulators on the observed subcellular toxicity of Pmel17 and A[unreadable] will be investigated using established proteotoxicity assays and the methodology outlined above. PUBLIC HEALTH RELEVANCE These investigations will lead to a clearer understanding of the innate cellular mechanisms for maintaining normal physiology with relationship to protein misfolding, enabling the development of new pharmaceuticals that will ameliorate some of the most important diseases of our era. [unreadable] [unreadable] [unreadable]